1. Field of the Invention
The field of this invention is the determination of intracellular calcium.
2. Related Art
The discovery that calcium plays a pleiotropic role in regulating cells in their response to ligands created an interest in being able to detect the level of calcium in cells in response to various agents, e.g. ligands, agonists and antagonists. Calcium plays a critical role in neuronal cells, muscle cells and other cells, where the cytosolic calcium acts as a regulator of cell expression. Because calcium is used by so many receptors and ion channels as a regulator of pathways leading to cellular function, measuring calcium can be a vicarious measure of the activity of a drug in regulating an ion channel or a receptor. Toward being able to measure cytosolic calcium, dyes have been developed that can enter a cell and fluoresce when bound to calcium. Measurement of the intracellular fluorescence provides a measure of the cytosolic calcium and, perforce, the activity of a compound in relation to a particular receptor. One can ensure that the target receptor is being measured, by using a competition between a known antagonist or introducing a gene(s) encoding a receptor or ion channel into a cell that would otherwise not respond to a drug by inducing a change in calcium concentration in the cytosol.
One problem in fluorescence measurement in biomedical assays is often that the fluorescence changes correlated with the biological cell action are small compared with the non-specific background fluorescence. As a result, the resolving power is greatly restricted. Conventional commercial measuring systems (fluorescence readers, Dynatech or SLT), cannot solve the problem, because owing to their optical measuring arrangement (excitation from ‘above’ through the fluorescent liquid column of the supernatant) the signal can barely be detected in comparison with the background.
Even very complicated measuring systems (NovelTech, FLIPR: Fluorescence Imaging Plate Reader) are only able to decrease this background fluorescence using a special laser illumination geometry (excitation below about 45°). Therefore, trying to solve the problem of background fluorescence by sophisticated instrumentation has not been entirely satisfactory.
In many cases of receptor or ion channel binding studies using fluorescently or luminescently labelled ligands, the labelled and unbound fraction in each case is removed by processes like washing. The washings introduce many uncertainties in the results, since cells can be subject to lysis, bound labeled ligand may be released, and unbound labeled ligand may be inadequately removed. There is, therefore, a need for a sensitive reliable methodology for measuring calcium without the introduction of methodologies that introduce uncertainties and variabilities in the results.
The subject invention provides improved sensitivity of optical analysis of fluorescently labeled or luminescent cells in a cellular assay in order to be able to measure, for example, membrane potential changes which are as low as possible on the basis of fluorescence changes of potential-sensitive dyes. In this case, the sensitivity of the measuring system should be sufficient that potential changes of below 5 mV can be detected at least qualitatively. In the case of luminescent cells, an increase in the detection of the luminescence signal should be achieved. Moreover, the method should be suitable for screening with a high sample throughput.
Relevant Literature
U.S. Pat. Nos. 6,420,183 and 6,221,612 describe cellular assays and methods for reducing background. Antibodies have been used in the past to quench fluorescent dyes that are spilled from cells upon cell lysis or other breaching of the cell membrane (U.S. Pat. No. 4,532,203). They have also been used to differentiate extracellular from internalized fluorescein labeled proteins (Sklar LA., J Cell Biochem 1982;20(2): 193-202; van Renswoude J, Proc Natl Acad Sci USA 1982 Oct;79(20):6186-90; and Schober JM, J Thromb Haemost. 2003 Nov;1(11):2404-10). Schnetcamp, et al. (1991) J. of Biol. Chem. 266, 22975-82 reports the use of antibodies in a calcium assay to remove background signal.